[CANCER RESEARCH 41, 1469-1482, April 1981) 0008-5472/81 /0041-OOOOS02.00 DMA-damaging Activity in Vivo and Bacterial Mutagenicity of Sixteen Hydrazine Derivatives as Related Quantitatively to their Carcinogenicity1 Silvio Parodi, Silvio De Flora, Marco Cavanna, Albiana Pino, Luigi Robbiano, Carlo Bennicelli, and Giovanni Brambilla2 Departments of Pharmacology [M. C.. A. P., L. P., G. B.¡,Hygiene [S. D. F., C. B.]. and Oncology [S. P.¡,Genoa University. 16132 Genoa, Italy ABSTRACT enees existing in the carcinogenic potency of different chemi cals, qualitative statements of positivity or negativity in a short- Sixteen hydrazine derivatives (hydrazine, 1,1-dimethylhydra- term test are of very limited value for an assessment of the zine, 1,2-dimethylhydrazine, phenylhydrazine, procarbazine, carcinogenic risk. isoniazid, isocarboxazid, nialamide, 2,4-dinitrophenylhydra- In this work, we attempted a quantitative comparison of the zine, phenelzine, hydralazine, dihydralazine, carbamylhydra- potencies of 16 hydrazine derivatives in: (a) inducing DNA zine, mebanazine, iproniazid, and 1-carbamyl-2-phenylhydra- fragmentation in liver and lung of male Swiss mice, as evaluated zine) were tested for DNA-damaging activity by the alkaline by alkaline elution technique; (b) inducing mutations in the elution technique and for mutagenic activity in the Salmonella- Sa/mone/te-microsome test, both in the presence and in the microsome (Ames) test. The first nine compounds listed (56%) absence of S-9 mix; and (c) inducing pulmonary tumors in were found to induce a significant DNA fragmentation in the chronically treated mice. The significance of a quantitative liver and/or in the lung of i.p.-treated male Swiss mice. The comparison is probably strongly dependent on the internal DNA-damaging potency varied over an ~30-fold range. Thir homogeneity of the sets of experiments that are compared. In teen of the first 14 compounds listed (81% of the total), this respect, the hydrazine derivatives appeared a rather con isocarboxazid being inactive, were positive in the Ames test, venient family of chemical compounds because their carcino with a broad range of activity towards the five bacterial strains genicity was studied mainly by a single group of investigators of Salmonella typhimurium used (TA1535, TA100, TA1537, according to a fixed protocol (42). Moreover, the carcinogenic TA1538, and TA98) and of metabolic behavior in the presence potencies of the hydrazine derivatives tested vary over a 1900- of S-9 mix containing rat liver, mouse liver, or mouse lung fold range, which seems to be a sufficiently large interval to postmitochondrial preparations from Aroclor-treated animals. allow for quantitative comparisons. The internal homogeneity The mutagenic potency varied over an almost 7000-fold range. of DNA damage and of mutagenicity data should be assured For 11 of the 16 hydrazine derivatives tested, homogeneous by the fact they were both directly obtained by our group in carcinogenicity data (induction of pulmonary tumors in mice experiments performed always according to the same experi chronically treated p.o.) were available from literature. Elabo mental protocol. Moreover, the 2 types of short-term tests allow ration of these data showed that carcinogenic potency varied for an additional interesting comparison: differences related to over an ~ 1900-fold range. The five most potent carcinogens in vitro versus in vivo metabolic activation. were all positive in the DNA damage test. Their carcinogenic potency varied over a 130-fold range and their DNA-damaging potency varied over a 22-fold range. DNA-damaging potency MATERIALS AND METHODS seemed to vary on a more compressed scale, but regression Chemicals analysis indicated the existence of a strong positive correlation between in vivo DNA-damaging and carcinogenic potencies, The name, structural formula, molecular weight, LD503 for while a lack of correlation was found between mutagenic and single i.p. administration in mice, source, and chemical purity carcinogenic potencies. There was no correlation between of the 16 hydrazine derivatives investigated are reported in DNA-damaging and mutagenic potencies. Table 1. Tetraethylammonium hydroxide was purchased from E. Merck, Darmstadt, West Germany; 3,5-diaminobenzoic acid INTRODUCTION dihydrochloride was obtained from Fluka AG, Buchs, Switzer land; dimethyl sulfoxide was purchased from BDH Chemicals, It has been repeatedly suggested that both induction of DNA Ltd., Poole, England. All other chemicals were reagent grade. damage in mammalian cells, measured as single-strand breaks (4, 10, 27, 30, 31) and mutagenicity in the Salmonella-micro- DNA Damage in 1/jVo-Alkaline Elution Assay some test (23, 24, 32, 33, 38) are, at least qualitatively, The in vivo assay of DNA damage was performed in non- correlated with carcinogenic activity. In recent years, however, it has become evident that, because of the tremendous differ- inbred male Swiss albino mice (aged 2 to 3 months) according to the following standardized protocol. Each hydrazine deriva 1 This work was supported by grants from the Consiglio Nazionale delle tive was tested at the following dosage schedules: (a) single Richerche (Progetto Finalizzato "Controllo della Crescita, Neoplastica"; Con i.p. administration of LD60 twice, reduced to LD60 (or one-half tracts 78.02856.96, 78.02803.96, and 79.00621.96). 2 To whom requests for reprints should be addressed, at Istituto di Farmacol of the LD50) if necessary because of lethal effect within 6 hr (or ogia dell'Università , Viale Benedetto XV, 2, 1-16132 Genoa, Italy. Received March 27. 1980; accepted October 16, 1980. 3 The abbreviation used is LDso, median lethal dose. APRIL 1981 1469 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1981 American Association for Cancer Research. 1Hydrazines testedStructural NameHydrazine M.W.H2N-NH2-H20formula purity98% hydrate 50.1 AG, Buchs, Switzer pure land 1,1-Dimethylhydrazine1 60.1CH3-NH-NH-CH3CH3x 12595' Fluka AG, Buchs, Switzer 95-98%pure98% landE. ,2-Dimethylhydrazine-2HCI -2HCI 133.0 Merck, Darmstadt, West pure Germany Carbamylhydrazine H2N-NH-CO-NH2 75.1 123170'SourceFlukaICN-K & K Laboratories, 95-99% pure Plainview, N. Y. PhenylhydrazineTable /=\ 108.1LO»"(mg/kg)156'Carlo Erba, Milan. ItalyChemical Reagent grade 1-Carbamyl-2-phenylhydrazine 151.2 198 ' Carlo Erba, Milan, Italy Reagent grade ¿ ^-NH-NH —CO-NH2 2,4-Oinitrophenylhydrazine 198.1 450' J. T. Baker Chemicals N. V., Reagent grade Deventer, Holland o-Phenylethylhydrazine oxalate 226.2 138' Imperial Chemical Indus 98% pure (mebanazine) CH-CH3 OXALATE tries, Cheshire, Great Britain /3-Phenylethylhydrazine •H2SO4 234.3 168 W. R. Warner & Co., Pharmaceutical grade (phenelzine) //-2- CH-NH-NH2 •H2S Eastleigh, Hampshire, Great Britain W-lsopropyl-a-<2- CO-NH-CH(CH3)2 257.8 800' Hoffmann-La Roche & Co., 98.5% pure methylhydrazino)-p-toluamide • Basel, Switzerland HCI (procarbazine) •HCI CH2- NH-NH—CH3 Isonicotinic acid hydrazide (isonia- 137.1 132 Vecchi & C. Piam. Genoa, 98% pure zid) Italy 0CO-NH—NH Isonicotinic acid 2-isopropylhydra- 179.2 640 Hoffmann-La Roche & Co., 99% pure zide (iproniazid) Basel, Switzerland 0CO-NH-NH-CHCCH3)2 Isonicotinic acid 2- 298.4 435 Pfizer Italiana, Rome, Italy Pharmaceutical grade [2(benzylcarbamoyl)ethyl]hydra- zide (nialamide) CO—NH-NH - (CH)- CO—NH-CH 5-Methyl-3-isoxazolecarboxylic 231.2 49 Hoffmann-La Roche & Co., 98.5% pure acid 2-benzylhydrazide (¡socar- Basel, Switzerland boxazid) 'CO—NH-NH — 1-Hydrazinophthalazine-HCI (hy- NH - NH, 196.7 83 Ciba-Geigy, Basel, Switzer- Pharmaceutical grade dralazine) land 1,4-Dihydrazinophthalazine •H2SO4 288.3 159 Ciba-Geigy, Basel, Switzer- Pharmaceutical grade (dihydralazine) land NH - NH, 3 LDso after single i.p. administration in mice. LDso's followed by asterisk were determined as described in "Materials and Methods" in male Swiss mice. All other LDso's were obtained from Ref. 34. 1470 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1981 American Association for Cancer Research. DNA Damage, Mutagenicity, and Carcinogenicity of Hydrazines 24 hr) from treatment; and (b) daily i.p. administration for 5 where V is the eluted volume in ml. As a first approximation, K (and sometimes 15) successive days of one-third of the LD50, is directly proportional to the number of single-strand breaks reduced to one-sixth of the LD50 or one-twelfth of the LD50 if (19). Controls were grouped into subsets of 20 experiments necessary because of lethal effects. each, which were carried out in a time interval of 3 to 4 weeks, The LDso's in mice for a single i.p. injection were as reported using the same batch of solutions for alkaline elution. The in the literature (34). When the LD50 was unknown, it was elution procedure remained unvaried for the whole period in determined at 10 days by administering i.p. to groups of 5 male which all the experiments were carried out; notwithstanding, mice for each dose a series of doses increasing serially by a some variability was observed among the various subsets of factor of 2; the LD50 was calculated (22) only after the doses controls. Consequently, the increase in DNA elution rate in given were sufficient to obtain both 0% and 100% mortality. duced by a given treatment and its statistical significance was Immediately before use, compounds soluble in water were calculated versus the subset of controls carried out approxi dissolved in 0.9% NaCI solution with small amounts of acid or mately in the same time interval. Because the distribution of alkali, if needed, to bring pH to ~7; compounds insoluble in the fractions of eluted DNA was not normal, the nonparametric water were suspended in 0.9% NaCI solution with 1% carbox- Wilcoxon 2-sample test was used for this statistical analysis ymethylcellulose as suspending agent. All compounds were (36). administered i.p. in 0.01 ml of vehicle per g of body weight. Tissue samples were taken for histológica! examination from Controls were given i.p.